A cosmid cloning vector is a plasmid which contains a lambda or lambdoid phage cos site, that is, the recognition site essential for packaging of linear concatenated DNA into lambda or lambdoid phage heads; a replicon, at least one selectable genetic marker; and at least one unique restriction endonuclease site in a nonessential region. These DNA sequences are herein referred to as "cosmid DNA".
Cosmid cloning vectors can be used to clone foreign DNA by inserting the foreign DNA into a restriction site in the cosmid DNA, packaging the recombinant DNA in infectious phage particles, preferably in vitro, and transfecting E. coli host cells. Once introduced into the host cells, the cosmids replicate in the manner of plasmids, due to the presence of the replicon. Transfected cells are selected based on a phenotypic trait contributed by the genetic marker, usually an antibiotic resistance marker.
Packaging is most efficient when the linear DNA to be packaged inside the phage head is about 23 to 31 Megadaltons (Md), that is, about 38 to 52 kilobase pairs (kb). Cosmid DNA can be considerably smaller than 52 kb. Thus, cosmid cloning vectors can be suitable for cloning large DNA fragments such as eukaryotic genes, which are typically interspersed in large sequences, and gene banks or libraries.
Hohn, Methods in Enzymology 68:299-309 (1979), and Collins, Methods in Enzymology 68:309-326 (1979), describe the morphogenesis of lambda phage particles and in vitro packaging of cosmid vectors. Several known cosmid vectors are described.
Collins and Hohn, Proc. Natl. Acad. Sci. U.S.A. 75(9):4242-4246 (1978), and Collins and Hohn, U.S. Pat. No. 4,304,863, disclose preparation of cosmids pJC703 and pJC720, 17.3 Md and 16 Md, respectively, each having a rifampicin resistance (Rif.sup.R) marker, and a cosmid of about 27.5 Md having Rif.sup.R and ampicillin resistance (Ap.sup.R) markers. The authors also disclose methods for in vitro packaging and transfection using cosmid cloning vectors containing foreign DNA.
Meyerowitz et al., Gene 11:271-282 (1980), describe construction of cosmids MUA-3, MUA-5 and MUA-10 and of eukaryotic gene libraries using MUA-3. The cosmids are about 3 Md and have tetracycline resistance (Tc.sup.R) markers.
Hohn and Collins, Gene 11:291-298 (1980), describe construction of cosmid pHC79, about 6.4 kb, having the two markers of plasmid pBR322, Ap.sup.R and Tc.sup.R.
At the Fifth Annual Mid-Atlantic Regional Conference on Extrachromosomal Genetic Elements, Oct. 9-11, 1981, Ocean City, Md., U.S.A., Taylor, the inventor of the cosmid cloning vector disclosed and claimed herein, reported cosmids pDPT5Cm and pDPT5Sp, each having two genetic markers: chloramphenicol resistance (Cm.sup.R) and Tc.sup.R in the case of pDPT5Cm, and streptomycin and spectinomycin resistance (Sm.sup.R /Sp.sup.R) and Tc.sup.R in the case of pDPT5Sp. An abstract of this presentation is published at Plasmid 8:100 (1982).
Hashimoto-Gotoh et al., Gene 16:227-235 (1981), describe construction of cosmid pHSG422, about 8.8 kb, having Ap.sup.R, Cm.sup.R and kanamycin resistance (Km.sup.R) markers.
Baldacci et al., Nucl. Acids. Res. 9:3575-3588 (1981), describe cosmids pFF1 and pFF2, having a Km.sup.R marker. The cosmid, pFF1, is claimed in French Patent No. 2,462,476 (Derwent Accession Number 33928D).
As stated previously, there is an upper limit of about 52 kb on the size of cosmid cloning vectors which can be packaged efficiently. Therefore, in order to clone large foreign DNA fragments, it is desirable to have cosmid cloning vectors in which the cosmid DNA, that is, the DNA carrying the cos site, a replicon, a marker and a unique restriction site, is small. Further, to facilitate selection of transfected cells containing the desired cosmid vector and for subsequent subcloning, it is desirable to have cosmid vectors containing two genetic markers with at least one of the markers having a unique restriction site such that insertion of foreign DNA into that site detectably affects the phenotype of transfected cells by insertional inactivation of the marker.